Mechanism Of CD147 In Regulating Rac1-mediated Cell Motility In Hepatocellular Carcinoma

The metastatic dissemination of tumors is the main cause of death for most cancer patients. Tumor metastasis is a highly complex and multistep process that requires a tumor cell to modulate its ability to adhere, invade into the surrounding extracellular matrix, migrate and proliferate at a secondary site. Migratory cancer cells undergo dramatic molecular and cellular changes by remodeling their cell-cell, cell-matrix adhesion, actin cytoskeleton and molecular processes involved in various signaling networks. Two different interconvertible modes of cell movement led by two unique signaling pathways have been demonstrated in individual tumor cells. Mesenchymal-type movement is characterized by an elongated cellular morphology that results from Rac-dependent actin assembly at the leading edge and requires extracellular proteolysis for Rac-dependent actin protrusions to be pushed through channels in the extracellular matrix. In contrast, amoeboid movement is characterized by a rounded, blebbing morphology that is independent of extracellular proteases. These two signaling pathways coordinate to regulate the cytoskeletal rearrangement that plays a critical role in the movement mode transition.CD147, which belongs to the immunoglobulin superfamily, is a glycosylated transmembrane protein that is highly expressed in various cancers. Overexpression of CD147 can enhance the metastatic potential of cancer cells due to increased adhesion, migration, invasion, and matrix metalloproteinases. Previously, we reported that the interaction between CD147 and Annexin Ⅱ can inhibit the amoeboid movement in hepatocellular carcinoma(HCC) cells. However, the mechanism of CD147 involved in mesenchymal movement is still unclear.The non-receptor tyrosine kinase Src is overexpressed and activated in many human malignancies. This kinase associates with many crucial aspects of tumor development and regulates a number of signaling pathways that are involved in survival, adhesion, invasion, migration and proliferation. In addition, deregulation of Src leads to constitutive kinase activation and cellular transformation. Decades of research have revealed the crucial role of Src in regulating small GTPases, and one of the major functions of Src is to modulate the actin cytoskeleton organization that controls cell migration. However, it remains unknown whether Src is involved in the interconversion between the two modes of individual cell movement and the underlying mechanisms are far from clear.This study is composed of the following two parts:1. CD147 promotes mesenchymal-type cell movement via FAK/Src pathway.We first investigated the effect of CD147 in regulating cell morphology and motility and the functional association between CD147 and Src in mesenchymal-type cell movement. We established a CD147 knockout SMMC-7721 cell line(K7721) to evaluate the role of CD147 in regulating cell morphology and motility. Results showed that overexpression of CD147 led to mesenchymal-type movement in HCC cells. Evidence indicated that the mesenchymal-type cell movement induced by CD147 was Src dependent, as observed by confocal microscopy and Rac1 activity assay. Furthermore, the phosphorylation of Src(p Y416-Src) could be up-regulated by CD147, and this regulation was mediated by focal adhesion kinase(FAK).2. The mechanism of Src-dependent activation of Rac1.The part I of this research has proved that CD147 promotes Src phosphorylation and mesenchymal-type movement in HCC cells. Rac1/WAVE signaling is critical for mesenchymal-type movment. Part Ⅱ aims to reveal the mechasims underlying Src mediated regulation of Rac1 signaling. Using si RNA screening of 11 members of DOCK family and Co-IP, we identified DOCK8 as an important GEF for Rac1. Furthermore, we found that Src could facilitate STAT3 phoshorylation and thus promoted STAT3 nucleus localization and target genes transcription. We also found that phosphor-STAT3 could promote DOCK8 expression and Rac1 activation.In summary, our results demonstrated that CD147 modulates Src activity through FAK when regulating cytoskeletal rearrangement and cell movement. We identified DOCK8 as a GEF for Rac1, which drives mesenchymal-type cell movement. Furthermore, Src promotes STAT3 phosphorylation and STAT3 facilitates DOCK8 transcription, thus enhancing DOCK8 expression and Rac1 activation. This study provides a novel mechanism of CD147 regulating mesenchymal-type movement in HCC cells.